High solubility composition with high isoflavone concentration and process of producing same

ABSTRACT

It is intended to provide a composition containing isoflavones which has a high purity and yet is highly soluble in water in a natural state without adding any solubilizers or subjecting to chemically modification. It is found out that a composition containing isoflavones with a high purity and a high solubility in water can be obtained by extracting soybean hypocotyls with a water-containing alcohol within a specific temperature range, then allowing a synthetic adsorbent resin to adsorb the obtained extract and eluting with a water-containing alcohol at a specific concentration.

TECHNICAL FIELD

The present invention relates to an isoflavone-containing compositionwhich is highly soluble in water although which has high purity, and aprocess for producing the composition.

BACKGROUND ART

Isoflavones found in soybeans mainly include free isoflavone glycosidessuch as daidzin, genistin and glycitin; malonyl isoflavone glycosidessuch as 6″-O-malonyl daidzin, 6″-O-malonyl genistin and 6″-O-malonylglycitin; acetyl isoflavone glycosides such as 6″-O-acetyl daidzin,6″-O-acetyl genistin and 6″-O-acetyl glycitin; and isoflavone aglyconessuch as daidzein, genistein and glycitein.

Isoflavones are active constituents of soybeans which have variousactivities such as alleviation of osteoporosis and climactericdisturbance, and have been widely used, for example, as an additive forhealth foods. Accordingly, various methods for extracting and purifyingisoflavones have been devised in order to efficiently ingestisoflavones.

JP-A 62-126186 (the patent document 1) discloses a method comprisingextracting soybean broth or soybeans with an aqueous organic solventunder reflux to obtain an extract, bringing the extract into contactwith a synthetic adsorbent resin such as a styrene-divinylbenzenepolymeric resin, and then eluting isoflavones from the resin with anaqueous organic solvent (for example, ethanol or 70% methanol). In thepatent document 1, although there is no detailed explanation about theconcentration of an aqueous organic solvent used for extraction, 80%aqueous ethanol is used in Example 2. However, the objective of thepatent document 1 is to obtain a large quantity of isoflavones at lowcost, and no attention is paid to the solubility of the resultingcomposition in water.

JP-A 8-283283 (the patent document 2) discloses a method for purifyingmalonyl isoflavone glycosides comprising extracting soybeans with waterto obtain an extract, bringing the extract into contact with a syntheticadsorbent resin, and then eluting the isoflavone glycosides from theresin with, for example, 20-50% aqueous ethanol. The patent document 2states that the suitable extraction temperature is 45 to 65° C. Theobjective of the patent document 2 is to purify malonyl isoflavoneglycosides that are relatively high hydrophilic. However, the extractionrate of the isoflavones is low because of extraction with water, andthus a composition containing a high concentration of isoflavones cannot be obtained. In addition, there is no description with respect tothe solubility of the resulting composition in water.

The compositions obtained by the methods described in the patentdocuments 1 and 2 contain a low concentration of isoflavones and do nothave satisfactory solubility in water.

JP-A 2002-80474 (the patent document 3) discloses a method for obtaininga free isoflavone glycoside fraction comprising extracting soybeanhypocotyls with aqueous lower alcohol (for example, 70% ethanol) at roomtemperature to 80° C. to obtain an extract, bringing the extract intocontact with an anion exchange resin having a uniform particle diameter,washing the resin with water, and then eluting free isoflavoneglycosides from the resin with, for example, 70% aqueous ethanol. Thecomposition obtained by purification with the anion exchange resincontains isoflavones at high concentration, namely, 60% or more.However, the composition is poorly soluble in water because it is apurified product of free isoflavone glycosides having a low solubility,and thus it has a problem of instability in an aqueous solution.

Accordingly, the methods described in the patent documents 1 to 3 cannot provide a composition containing a high concentration of isoflavonesand having a high solubility, which can be added into beverage.

Such problem arises from the low solubility of isoflavones themselves.For example, the solubility of daidzin is only about 3.3 mg/100 ml. JP-A10-298175 (the patent document 4) discloses a method for improving thesolubility of isoflavones in water which comprises extracting soybeanhypocotyls with 70 to 90% aqueous alcohol at about 70° C. under refluxto obtain an extract, bringing the extract into contact with a syntheticadsorbent resin, eluting the resin with 30 to 80% aqueous alcohol toobtain an isoflavone-containing composition, and then forming aninclusion compound of the isoflavone-containing composition withcyclodextrin. Since this method requires an addition of at least anequimolar amount of cyclodextrin to the isoflavone extract, theisoflavone content in the final product is extremely decreased.Moreover, since the solubility of the final product depends on thesolubility of cyclodextrin itself, a step of dissolving the inclusioncompound by heating is needed when it is used, and further, it can notbe dissolved at a higher concentration.

JP-A 2000-327692 (the patent document 5) discloses a method forincreasing the solubility comprising formation of α-glycosyl isoflavonesby an action of glycosyltransferase in the presence of an α-glycosylsaccharide compound. However, this method also has a problem of decreasein the isoflavone content because it requires an addition of at least anequimolar amount of the saccharide compound.

Accordingly, there has been a need for development of anisoflavone-containing composition which has high purity and can bestably dissolved in water at a high concentration in a natural statewithout using any solubilizing agents such as cyclodextrin or anyadditives such as saccharide compounds and thus is suitable to use inbeverage and the like.

REFERENCES

The patent document 1: JP-A 62-126186

The patent document 2: JP-A 8-283283

The patent document 3: JP-A 2002-80474

The patent document 4: JP-A 10-298175

The patent document 5: JP-A 2000-327692

DISCLOSURE OF INVENTION

Problem to be Solved by the Invention:

The present invention is to provide a novel isoflavone-containingcomposition which has high solubility in water although which has highpurity in a natural state without adding any solubilizing agents orwithout applying any chemical modifications, and a process for producingthe isoflavone-containing composition.

Solution for the Problem:

The present inventors studied intensively and as a result, they preparedan isoflavone-containing composition containing malonyl isoflavoneglycosides, isoflavones other than malonyl isoflavone glycosides andsaponins in specified proportions from soybean hypocotyls, and thenfound that the isoflavone-containing composition surprisingly containeda high concentration of isoflavones and had remarkably high solubility.In addition, the present inventors found that the isoflavone-containingcomposition could be obtained by extracting soybean hypocotyls withaqueous alcohol within a specified temperature range, allowing theextract to be adsorbed to a nonpolar synthetic adsorbent resin, and theneluting the resin with aqueous alcohol having a specified alcoholconcentration, as a particularly preferable process.

That is, the present invention discloses the following inventions:

(1) an isoflavone-containing composition which comprises 15 to 95% byweight of malonyl isoflavone glycosides, 0 to 50% by weight ofisoflavones other than malonyl isoflavone glycosides and 5 to 60% byweight of saponins, by taking the sum of the total amounts ofisoflavones and saponins in said composition as 100% by weight;

(2) the isoflavone-containing composition according to the above (1),which is prepared from soybean hypocotyls as the starting material;

(3) the isoflavone-containing composition according to the above (1),wherein the proportion of group A saponins in saponins is 55% by weightor more, by taking the total amount of saponins in said composition as100% by weight;

(4) the isoflavone-containing composition according to the above (1),which has a water solubility of at least 20 mg/100 ml at 25° C. based onthe amount of isoflavones;

(5) a process for producing the isoflavone-containing compositionaccording to the above (2), which comprises the step of extractingsoybean hypocotyls with 15 to 95% by volume aqueous ethanol at 10 to 50°C.;

(6) a process for producing the isoflavone-containing compositionaccording to the above (2), which comprises the steps of:

(A) extracting soybean hypocotyls with 15 to 95% by volume aqueousethanol at 10 to 50° C. to obtain an extract;

(B) bringing a solution of the extract obtained by the step (A) in waterinto contact with a nonpolar adsorbent resin to allow isoflavones to beadsorbed to the resin; and

(C) eluting isoflavones from the adsorbent resin with 15 to 40% byvolume aqueous ethanol;

(7) an edible composition containing the isoflavone-containingcomposition according to the above (1); and

(8) a process for fractionating isoflavones and saponins, whichcomprises extracting soybean hypocotyls with 15 to 95% by volume aqueousethanol at 10 to 50° C. to obtain an extract, bringing a solution of theextract in water into contact with a nonpolar adsorbent resin, elutingan isoflavone-containing fraction from the adsorbent resin with 15 to40% by volume aqueous ethanol, and then eluting a saponin-containingfraction from the adsorbent resin with 65 to 90% by volume aqueousethanol.

EFFECTS OF THE INVENTION

The present invention can provide an isoflavone-containing compositionwhich has high solubility and contains a high concentration ofisoflavones in a natural state without adding any solubilizing agents orwithout applying any chemical modifications, and said composition ishighly soluble in water as compared with a conventionalisoflavone-containing composition that has been considered to be readilysoluble in water.

BEST MODE FOR CARRYING OUT THE INVENTION

The isoflavone-containing composition of the present inventionpreferably contains isoflavones in an amount of 25 to 95% by weight,more preferably 25 to 60% by weight, still more preferably 30 to 60% byweight, most preferably 40 to 60% by weight of the total solid content.

The isoflavone-containing composition of the present invention ischaracterized by the following three points, wherein the sum of thetotal amounts of isoflavones and saponins contained in said compositionis defined as 100%:

1. the content of malonyl isoflavone glycosides is 15 to 95% by weight,more preferably 20 to 90% by weight, still more preferably 25 to 85% byweight, most preferably 30 to 80% by weight;

2. the content of isoflavones other than malonyl isoflavone glycosides(e.g., isoflavone glycosides, acetyl isoflavone glycosides, isoflavoneaglycones and the like, i.e., the amount after subtracting the contentof malonyl isoflavone glycosides from the total content of allisoflavones) is 0 to 50% by weight, more preferably 0 to 45% by weight,still more preferably 0 to 40% by weight, most preferably 0 to 30% byweight; and

3. the content of saponins is 5 to 60% by weight, more preferably 10 to50% by weight, still more preferably 15 to 45% by weight, mostpreferably 20 to 40% by weight.

By using component percentages within the above-described ranges, anisoflavone-containing composition can be prepared, which has highsolubility in water although which contains a high concentration ofisoflavones, and which maintains high stability even under cold storageafter dissolved.

The component percentages of the isoflavone-containing composition ofthe present invention are not particularly limited so long as they arewithin the above-described ranges. More specifically, theisoflavone-containing composition of the present invention may contain,in addition to isoflavones and saponins, 8 to 20% by weight of proteinsbased on the total solid content and may further contain saccharides. Itis more preferable that the content of saponins is ¼ to ¾ times byweight the content of isoflavones.

Soybean saponins comprise group A saponins, group B saponins, group Esaponins and the like. The appropriate proportion of group A saponins insaponins contained in the isoflavone-containing composition of thepresent invention is at least 55% by weight, more preferably at least60% by weight, by taking the total amount of saponins in saidcomposition as 100% by weight.

In the isoflavone-containing composition of the present invention, thesolubility of isoflavones in water at 25° C. is at least 20 mg/100 ml,preferably 50 to 1200 mg/100 ml, more preferably 100 to 1200 mg/100 ml,still more preferably 500 to 1200 mg/100 ml. The isoflavone-containingcomposition of the present invention is not only highly soluble, but itshigh concentration solution as described above also does not formprecipitates and become turbid even under cold storage (10° C. or less)for a long period. In the present invention, “solubility” is defined asthe maximum amount (g) of a solute that can dissolve in 100 ml of waterat 25° C., and expressed as the amount (mg) of isoflavones.

The isoflavone-containing composition of the present invention maycontain a solubilizing agent such as cyclodextrin or starchoctanylsuccinate to solubilize insoluble substances, or may contain ahydrophilic saccharide compound to allow binding of the saccharidecompound with isoflavones in the composition by the action ofglycosyltransferase or the like. However, addition of such additives isnot essential and it is preferable to avoid adding such additives. Thisis because the proportion of isoflavones in the total solid content ofthe resulting composition is decreased when such additives are added,and the resulting composition hardly dissolves unless it is heated dueto the low solubility of cyclodextrin at room temperature when thecomposition contains cyclodextrin. Accordingly, if a solubilizeing agentis added, it is preferable that the amount added is less than the amountof isoflavones contained in the isoflavone-containing composition.

The isoflavone-containing composition of the present invention describedabove is a composition containing a high concentration of isoflavonesand having high solubility, and is also a novel natural material thatrequires no addition of any solubilizing agents and no chemicalmodifications. It has not been elucidated yet a reason why theisoflavone-containing composition of the present invention has highsolubility in spite of the fact that the composition contains a highconcentration of isoflavones that are naturally poorly soluble in water.In other words, the solubilizing mechanism has not been elucidated yet.However, it seems that the coexistence of a specified amount ofcomponents other than saponins and isoflavones contained in soybeanhypocotyls result in some interaction with isoflavones and therebyisoflavones become highly soluble.

Aspects of a process for producing the isoflavone-containing compositionof the present invention will be described hereinafter.

(Starting Material for Extraction)

The starting material for extracting the isoflavone-containingcomposition of the present invention is not limited and includes wholesoybean, defatted soybean, isolated soybean protein, soybean curd refuse(okara) and the like. Preferably soybean hypocotyls are used. Theisoflavone content of soybean hypocotyls is 1 to 2% by weight of thetotal solid content, which is 10 times or more as much as that ofsoybean seeds. Thus, soybean hypocotyls are a preferable material forobtaining an isoflavone-containing composition with high purity, and arealso important for obtaining an isoflavone-containing composition withhigh solubility. Although the relation between solubility and saponincomposition is unknown, the proportion of group A saponins in the totalsaponins of soybean hypocotyls is 60% by weight or more and theproportion of group B saponins is lower. Such saponin composition (theratio of group A:group B) in soybean hypocotyls is different from thatin soybean cotyledons. A method for preparing soybean hypocotyls is notparticularly limited and for example, they may be readily separated byremoving the surface skins of soybeans and then roughly crushing them.Preferably, the soybean hypocotyls thus obtained may be sieved toenhance the purity of hypocotyls. Alternatively, commercially availableproducts sold as “soybean hypocotyls” or “soybean germs” may be used.Soybean hypocotyls may be subjected to a pretreatment such as a heatingtreatment for the purpose of improving the flavor or the like. However,malonyl isoflavone glycosides may be converted into free glycosides byheating, and in this case, it is difficult to separate isoflavones fromother components such as saponins. Therefore, it is preferable to useraw soybean hypocotyls or soybean hypocotyls after a slight heattreatment. Full-fat soybeans or defatted soybeans may be also used.However, they have a low isoflavone content, so that it costs time andmoney to attain the desired purity of isoflavone. Therefore, soybeanhypocotyls are preferred.

(Extraction Solvent)

As a solvent for extracting isoflavones from soybean hypocotyls, anaqueous alcoholic organic solvent such as ethanol, methanol or propanolmay be used. In view of use in foods, ethanol is preferably used. Thealcohol concentration of an extraction solvent is preferably 15 to 95%by volume, more preferably 30 to 90% by volume, still more preferably 40to 85% by volume, and most preferably 60 to 80% by volume. When thealcohol concentration is too low, isoflavones are difficult to extractand the yield of isoflavones decreases, and in addition, isoflavoneglycosides such as daidzin and genistin and malonyl isoflavoneglycosides tend to be converted into isoflavone aglycones such asdaidzein and genistein during extraction by the effect of β-glucosidasethat is naturally contained in soybean hypocotyls, so that it isdifficult to obtain an isoflavone-containing composition with highsolubility. When the alcohol concentration is too high, the yield ofisoflavones decreases and in addition, the production cost increases.Therefore, an alcohol solution with such high concentration isinappropriate.

(Extraction Temperature)

In the present invention, it is important that the extract temperatureis 10 to 50° C., more preferably 20 to 40° C. It is needless to say thatextraction may be performed at room temperature as long as the roomtemperature is within the above-described temperature range. When theextraction temperature is too low, the extraction efficiency decreases.On the contrary, when the extraction temperature is too high, malonylisoflavone glycosides are thermally decomposed and then converted intofree isoflavone glycosides, so that it is difficult to obtain anisoflavone-containing composition with high solubility at 25° C.Isoflavones in the form of solution containing isoflavones at highconcentration can be recovered from hypocotyls as the starting materialin a high yield of 80% or more by extraction with the specifiedconcentration of a solvent and at the specified temperature as describedabove, depending on extraction conditions. The extraction step describedabove is preferably repeated more than once to enhance the yield ofisoflavones.

(Adsorbent Treatment)

An isoflavone-containing composition having high solubility andcontaining a high concentration of isoflavones is purified from thecrude extract of isoflavones obtained by the extraction step describedabove. A first purification step comprises dissolving the crude extractin water, if necessary, and bringing the crude extract into contact witha nonpolar adsorbent resin. Since the extract obtained by the extractionstep described above contains alcohol, it is preferably subjected topretreatment such as alcohol removal and concentration, if necessary.Since lipids contained in the starting material may be extracted whenthe alcohol concentration of an extraction solvent is high, the startingmaterial or the extract may be defatted. A nonpolar adsorbent resin tobe used includes preferably nonpolar synthetic adsorbent resins such asHP-20 (manufactured by Mitsubishi Chemical Co.), SP-825 (manufactured byMitsubishi Chemical Co.), Amberlite XAD-2 and KAD-4 (manufactured byRohm and Haas Co.), and Duolite S-861 and S-862 (manufactured bySumitomo Chemical Industry Co.), which are porous styrene-divinylbenzeneresins. Duolite C26A (manufactured by Sumitomo Chemical Industry Co.),which is a polar cation exchange resin, and WA-30 (manufactured byMitsubishi Chemical Co.), which is an anion exchange resin, are notsuitable because they do not adsorb isoflavones or the resultingisoflavone material is low soluble. This adsorbent treatment may beachieved batch-wise by putting the adsorbent rein in a tank, or may beachieved by filling a column with the adsorbent resin. It is preferableto allow a solution of the crude extract in water to pass through aresin column filled with the resin, from the viewpoint of productionefficiency and productivity.

(Elution of Isoflavone-containing Composition)

Next, a specified isoflavone is selectively eluted and collected from afraction adsorbed to the adsorbent resin with an aqueous alcoholicorganic solvent. The aqueous alcoholic organic solvent is preferablyaqueous ethanol. In the present invention, it is important that theethanol concentration of an elution solvent is 15 to 40% by volume, morepreferably 20 to 35% by volume. When the ethanol concentration is toolow, it requires a large volume of the solvent to collect anisoflavone-containing composition and isoflavones are poorly eluted, sothat the isoflavone concentration in the resulting compositiondecreases. When the ethanol concentration is too high, a large quantityof impurities other than isoflavones such as saponins is eluted, so thatthe isoflavone purity in the resulting composition decreases, and inaddition, nonpolar substances with low-water solubility such asisoflavone aglycones are also eluted, so that an isoflavone fractionwith high solubility can not be obtained.

(Processing of Isoflavone-containing Composition)

After the isoflavone-containing composition obtained by the productionmethod described above is heated, evaporated under reduced pressure toremove the organic solvent and then concentrated, the concentrate may beused as it is as an “isoflavone extract” or may be powdered by spraydrying, freeze drying or the like. The isoflavone-containing compositionthus obtained is a novel isoflavone-containing composition containing ahigh concentration of isoflavones and having extreme high solubility,wherein the isoflavone content is 25 to 95% by weight of the total solidcontent and the water solubility at 25° C. is at least 20 mg/100 mlbased on the amount of isoflavones.

(Acid Precipitation Treatment)

The isoflavone-containing composition obtained by the above method canbe used as it is within the concentration range usually used, forexample, in 20 mg/100 ml based on the amount of isoflavones. However,when the isoflavone-containing composition is added to acidic food orbeverage in high concentration, the isoflavone-containing compositionmay be previously subjected to pH adjustment to the acid range followedby removal of insoluble substances generated (for example, proteins) toenhance the transparency of the solution. Edible inorganic acid ororganic acid may be used for the pH adjustment, and for example,hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, malicacid, citric acid or ascorbic acid may be used. The pH of theisoflavone-containing composition may be adjusted to the pH or lower offood or beverage to which the composition is added, usually pH 4 orless, preferably pH 3.5 or less. Then, the isoflavone-containingcomposition may be maintained at the acidic pH for a given period oftime. During all that time, the composition is maintained suitably atroom temperature or below, preferably at 10° C. or below. An extract ofsoybean hypocotyls or the like usually contains proteins as well asisoflavones. When the proteins in the extract are precipitated underacidic conditions, their precipitation tends to be accompanied byprecipitation of isoflavones (coprecipitation). In the presentinvention, however, the coprecipitation is prevented because the proteincontent of the isoflavone-containing composition of the presentinvention is decreased by extraction with an alcohol and purificationwith an adsorbent resin. Isoflavones may be separated from insolublesubstances by a conventional solid-liquid separation method such asfiltration and centrifugation.

(Elution of Saponin-containing Fraction)

In the present invention, after eluting an isoflavone-containingfraction from a nonpolar adsorbent resin with 15 to 40% by volumeaqueous ethanol, a fraction mainly comprising saponins remains adsorbedto the resin. The saponin-containing fraction can be eluted with ahigher concentration of alcohol, preferably 60 to 95% by volume, morepreferably 65 to 90% by volume aqueous ethanol to obtain asaponin-containing composition containing saponins in an amount of 40%by weight or more of the total solid content. As described above, anisoflavone fraction containing a high concentration of isoflavones andhaving high solubility, and a saponin fraction containing a highconcentration of saponins can be efficiently separated by obtaining anextract at the specified temperature, allowing the extract to beadsorbed to a nonpolar adsorbent resin and then eluting the fractionsfrom the resin with different concentrations of aqueous alcoholsolutions, and thereby a saponin material containing a highconcentration of saponins can be provided.

(Applicability to Foods)

By adding only a small amount of the isoflavone-containing compositionthus obtained to all kinds of edible compositions such as medicines,foods and beverages, a large amount of isoflavones can be provided tothose who ingest said edible compositions, because theisoflavone-containing composition contains a high concentration ofisoflavones. Moreover, since saponins are contained together in theisoflavone-containing composition, both ingredients of isoflavones andsaponins can be effectively ingested. Since the isoflavone-containingcomposition is highly soluble, it is particularly effective in liquidmedicines and foods, and beverages. The isoflavone-containingcomposition can be provided in solid form such as a tablet or a granule.The isoflavone-containing composition can be also provided inconcentrate form such as a portion by taking advantage of its highsolubility, and therefore can be used as an ingredient of seasoningssuch as jams and sauces.

In the present invention, analysis of isoflavones was performedaccording to a standard method for analysis of soybean isoflavone foodsby Japan Health Food and Nutrition Food Association. Saponins werequantified by thin layer chromatography as described hereinbelow, andproteins were quantified by Kjeldahl method.

(Quantification Method of Isoflavone)

A sample corresponding to 1 to 10 mg of isoflavones was preciselyweighed, and 25 mL of 70% (v/v) ethanol was added to the sample. Afterextraction with stirring for 30 minutes at room temperature, the extractwas centrifuged. The residue was further extracted twice in the same wayas in the above. Extract solutions from three extractions in total werecombined, adjusted to a volume of 100 mL with 70% (v/v) ethanol, andthen filtered with a 0.45 μm PVDF filter to obtain a test solution. In aconfirmatory test of isoflavones, the peaks of the test solution wereconfirmed at about the same retention times as compared with 12 kinds ofstandard samples, namely, daidzin, genistin, glycitin, daidzein,genistein, glycitein, malonyl daidzin, malonyl genistin, malonylglycitin, acetyl daidzin, acetyl genistin and acetyl glycitin (allmanufactured by Waco Pure Chemical Industries, Inc.). In a quantitativetest, the concentrations of 12 isoflavones in the test solution werequantified (as equivalent converted into daidzin) using daidzin standardsamples as standard. The true isoflavone concentrations were calculatedby multiplying the obtained values by the following quantificationconstants: daidzin (1.000), genistin (0.814), glycitin (1.090), malonyldaidzin (1.444), malonyl genistin (1.095), malonyl glycitin (1.351),acetyl daidzin (1.094), acetyl genistin (1.064), acetyl glycitin(1.197), daidzein (0.583), genistein (0.528), and glycitein (0.740). Thetotal amount of isoflavones was determined from the sum of theconcentrations of all isoflavones. HPLC conditions of the test solutionsand the standard solutions are shown in Table 1.

TABLE 1 HPLC Conditions (Column) YMC-Pack ODS-AM-303 (4.6 × 250 mm)(Mobile phase) (Solution A) acetonitrile:water:acetic acid = 15:85:0.1(v/v/v) (Solution B) acetonitrile:water:acetic acid = 35:65:0.1 (v/v/v)(Solution A → solution B) linear concentration gradient (50 minutes)(Flow rate) 1.0 mL/min (Temperature) 25° C. (Detection) UV 254 nm(Injection volume) 10 μL(Quantification of Saponin)

A sample was precisely weighed, extracted with stirring for 1 hour withmethanol, and then centrifuged to obtain an extract solution. Thisprocedure was repeated again. The resulting extract solutions werecombined, and the volume of the combined solution was adjusted to agiven volume. This sample solution was spotted on a thin layerchromatography (TLC) plate. The Rf value of the sample spot was comparedwith that of a saponin standard to confirm that there was the saponinspot on the sample. An integration value of the spot area was determinedusing a standard line previously prepared by using a saponin standard,and thereby the amount of saponins was calculated. The conditions ofthin layer chromatography are shown in Table 2.

TABLE 2 TLC conditions (TLC plate) Silica gel plate 60F254 (thickness0.25 mm, manufactured by Merck Co.) (Developmentchloroform:methanol:water solvent) (bottom layer of 65:25:10 (v/v/v)solution) (Development) 18 cm (Detection) heating at 105° C. for 15minutes after spraying 10% sulfuric acid solution

EXAMPLES

Hereinafter, the present invention is illustrated by reference toExamples which do not restrict the technical scope of the presentinvention.

Example 1

To 500 g of raw soybean hypocotyls was added 2.0 L of 70% by volumeaqueous ethanol, and the mixture was extracted with stirring at 30° C.for 8 hours. After the extract solution was separated by filtration, theresidue was extracted again with 2.0 L of 70% by volume aqueous ethanolin the same way as in the above. The two extract solutions werecombined.

The combined extract solution was concentrated under reduced pressure at40° C. to completely remove the organic solvent. The crude extract thusobtained was dissolved in water, and applied at SV2 onto a column (100mL) filled with a nonpolar synthetic adsorbent resin, Diaion HP-20(manufactured by Mitsubishi Chemical Co.). Then, the column was elutedwith 30% by volume aqueous ethanol to obtain an isoflavone fraction. Thefraction was concentrated under reduced pressure at 40° C., dried andpowdered to obtain 15 g of an isoflavone-containing composition. Theconcentration of isoflavones in the obtained isoflavone-containingcomposition was 48.0% based on the weight of the total solid content.The composition also contained 23.8% of saponins and 14.1% of proteins.

Experiment Example 1

Investigation of the Concentration of Ethanol in an Extraction Solvent

Soybean hypocotyls were subjected to extraction in the same way as inExample 1 except that the ethanol concentration of an extraction solventwas changed as shown in Table 3, and changes in the relative proportionsof isoflavones in the resulting extract and in the yield of isoflavonesfrom soybean hypocotyls were examined. The results are shown in Table 3.

TABLE 3 Relative proportions of isoflavones in ethanol extract solutionsand yields of isoflavones Ethanol concentration (% by volume) 10 20 4050 70 90 100 Mal-ISO 18.0 65.3 60.3 59.6 55.3 45.3 28.6 Ac-ISO 6.3 2.16.3 4.1 3.3 0.0 0.0 ISO 23.7 20.6 31.1 33.0 36.3 46.6 64.3 Agl 52.0 12.02.3 3.3 5.1 8.1 7.1 Yield of 63 76 82 82 86 38 10 isoflavones (%) *Mal-ISO: malonyl isoflavone glycoside Ac-ISO: acetyl isoflavoneglycoside ISO: isoflavone glycoside Agl: isoflavone aglycone

When the ethanol concentration of an extract solvent was 10% by volume,the yield of isoflavones was decreased, the content of isoflavoneaglycones was increased, and the content of malonyl isoflavoneglycosides was rapidly decreased. This is probably because β-glucosidasein soybean hypocotyls reacted with malonyl isoflavone glycosides due tothe low concentration of ethanol and thereby the malonyl isoflavoneglycosides were decomposed to aglycones. When the ethanol concentrationwas 100% by volume, the yield of isoflavones was rapidly decreased, thecontent of isoflavone glycosides was increased, and the content ofmalonyl isoflavone glycosides was rapidly decreased. Accordingly, theconcentration of ethanol in an extraction solvent is suitably at least15 to 95% by volume, more preferably 30 to 90% by volume, morepreferably 40 to 85% by volume, most preferably 60 to 80% by volume.

Comparative Example 1

Extraction with Ethanol at High Temperature

To 500 g of raw soybean hypocotyls was added 1.5 L of 70% by volumeaqueous ethanol, and the mixture was extracted with heating under refluxat 70° C. for 8 hours. After the extract solution was separated byfiltration, the residue was extracted under reflux again with 1.5 L of70% by volume aqueous ethanol in the same way as in the above. The twoextraction solutions were combined.

The combined extract solution was concentrated under reduced pressure at60° C. to completely remove the organic solvent. The extract thusobtained was dissolved in water, and applied at SV2 onto a column (100mL) filled with a nonpolar adsorbent resin, HP-20 (manufactured byMitsubishi Chemical Co.). The column was eluted with 30% by volumeaqueous ethanol to obtain an isoflavone fraction. The fraction wasconcentrated under reduced pressure at 60° C., dried and powdered toobtain 20 g of an isoflavone-containing composition. The isoflavoneconcentration of the obtained isoflavone-containing composition was 38%based on the weight of the total solid content. The saponinconcentration was 18% by weight of the total solid content.

Comparative Example 2

Extraction with High Concentration of Ethanol

An isoflavone fraction was obtained in the same way as in Example 1except that the concentration of aqueous ethanol used for elution ofisoflavones from a column was 70% by volume. The fraction wasconcentrated under reduced pressure at 60° C., dried and powdered toobtain 30 g of an isoflavone-containing composition. The isoflavoneconcentration of the obtained isoflavone-containing composition was20.7% by weight of the total solid content. The saponin concentrationwas 33% by weight of the total solid content.

Comparative Example 3

Extraction at High Temperature, Anion Resin, and Elution with HighConcentration of Ethanol

To 500 g of raw soybean hypocotyls was added 1.5 L of 70% by volumeaqueous ethanol, and the mixture was extracted with heating under refluxat 70° C. for 8 hours. After the extract solution was separated byfiltration, the residue was extracted under reflux again with 1.5 L of70% by volume aqueous ethanol in the same way as in the above. The twoextract solutions were combined.

The combined extract solution was concentrated under reduced pressure at60° C. to completely remove the organic solvent. The extract thusobtained was dissolved in water, and applied at SV2 onto a column (100mL) filled with an anion exchange resin, WA-30 (manufactured byMitsubishi Chemical Co.). Then, the column was eluted with 70% by volumeaqueous ethanol to obtain an isoflavone fraction. The fraction wasconcentrated under reduced pressure at 60° C., dried and powdered toobtain 6 g of an isoflavone-containing composition. The isoflavoneconcentration of the obtained isoflavone-containing composition was 75%based on the weight of the total solid content. The saponinconcentration was 0.5% by weight of the total solid content.

The relative proportions of isoflavones in the isoflavone-containingcompositions obtained in Example 1 and Comparative Examples 1 to 3 areshown in Table 4. The isoflavone-containing composition obtained inExample 1 had a high proportion of malonyl isoflavone glycosides and lowproportions of isoflavone glycosides and isoflavone aglycones, ascompared with those in the isoflavone-containing compositions obtainedin Comparative Examples 1 to 3. The reason why the proportion of malonylisoflavone glycosides was decreased in Comparative Example 1 wasprobably that malonyl isoflavone glycosides were decomposed toisoflavone glycosides by raising the extraction temperature to 70° C.The reason why the proportion of malonylisoflavone glycosides wasdecreased in Comparative Example 2 was probably that a large amount ofcomponents other than malonyl isoflavone glycosides were eluted byincreasing the concentration of ethanol used for elution.

TABLE 4 (Unit: %) Comp. Comp. Comp. Example 1 Example 1 Example 2Example 3 (condition) Ethanol Concentration 70% 70% 70% 70% forExtraction Ethanol Concentration 30% 30% 70% 70% for Elution Extraction30° C. 70° C. 30° C. 70° C. Temperature Kind of Resin Nonpolar NonpolarNonpolar Anion- adsorbent adsorbent adsorbent exchange (Relativeproportions of Isoflavones) Malonyl glycoside 68.3 19.8 30.3 0.3 Acetylglycoside  8.8 7.6 5.5 0.9 Free glycoside 22.8 67.6 53.8 98.5 Aglycone 0.1 5.0 10.4 0.3

Comparative Example 4

Preparation of Soluble Isoflavones Using Cyclodextrin

In 500 ml of 50% aqueous ethanol at 80° C., 5 g of theisoflavone-containing composition obtained in Comparative Example 1(extraction with high concentration of ethanol) and 25 g ofβ-cyclodextrin (manufactured by Nihon Shokuhin Kako Co.) were dissolved,and the solution was stirred at 80° C. for 3 hours. The solution wasthen left at room temperature overnight. The supernatant was dried underreduced pressure to obtain 10 g of powder. The concentration ofisoflavones in the isoflavone-containing composition thus obtained was8.4% based on the weight of 15 the total solid content. Theconcentration of saponins was 1.8% by weight of the total solid content.

Experiment Example 2

The isoflavone-containing compositions obtained in Example 1 andComparative Examples 1 to 4 were dissolved in water at pH 7.0 at 25° C.with stirring so that the isoflavone concentration is 20 mg/100 ml.After stirring, the solubility was evaluated. The stability after coldstorage at 4° C. for 2 weeks was also evaluated. The results are shownin Table 5.

TABLE 5 Example Comparative Example 1 1 2 3 4 Ethanol Concentration for70 70 70 70 70 Extraction (%) Extraction Temperature 30 70 30 70 30 (°C.) Ethanol Concentration for 30 30 70 70 70 Elution (%) IsoflavoneContent (dry %) 48 38 20.7 75 8.4 Malonyl isoflavone 32.8 7.5 6.3 0.20.7 glycoside Content (dry %) Saponin Concentration 24 18 33 0.5 1.8(dry %) Solubility after ∘ x x x x stirring (25° C.) Stability after 2 ∘x x x x weeks' cold storage (Evaluation criteria) ∘: no precipitate andno turbidity were observed. x: precipitates or turbidity was observed.

As shown in Table 5, in Example 1, the isoflavone percentage in thetotal solid content was as high as 48% and the solubility at 25° C. wasalso extremely high. In Example 1, the stability after cold storage for2 weeks was also extremely good. Therefore, an isoflavone-containingcomposition having a high concentration of isoflavones and havingextreme high water solubility could be obtained under the conditions ofExample 1. The maximum amount (solubility) of the isoflavone-containingcomposition obtained in Example 1 that could dissolve in water at 25° C.was surprisingly at least 1,000 mg/100 ml based on the amount ofisoflavones.

In Comparative Example 1, although the isoflavone content was as high as38%, both of the solubility at 25° C. and the stability after coldstorage were poor. This was probably because naturally water-insolublesubstances including isoflavone aglycones, isoflavone glycosides andpigments were extracted as a result of extraction with heating underreflux at a high temperature (70° C.).

In Comparative Example 2, the isoflavone content was as low as 20% andthe solubility at 25° C. was also poor. This was probably because alarge amount of poorly water-soluble nonpolar substances was eluted fromthe adsorbent resin by using a high concentration of aqueous ethanol forelution of an isoflavone fraction.

Although the isoflavone content was as high as 75% in theisoflavone-containing composition obtained in Comparative Example 3, thecomposition did not dissolve at all in water at 25° C. This was probablybecause the solubility of isoflavones itself was naturally as low asabout 5 mg/L and could not be improved by high degree of purification.

In Comparative Example 4, the isoflavone content was as low as 8.4%because the percentage of saccharides in the total solid content wasincreased as a result of inclusion of isoflavones with cyclodextrin. Theobtained isoflavone-containing composition did not dissolve at all inwater at 25° C. This was probably because the solubility of the obtainedcomposition depended on that of cyclodextrin and cyclodextrin did notdissolve in water at 25° C. In order to dissolve the composition,heating at 80° C. for 10 minutes or more was needed.

As seen from the above-described results, only in Example 1, that is, inthe case of combining extraction and elution with the specifiedconcentration of ethanol, and extraction at the specified temperature,an isoflavone-containing composition having high purity, high solubilityand good stability under cold storage could be obtained. The relativeproportions of components of the composition having such excellentproperties were examined. As a result, it has been found that it isimportant that: (1) the content of isoflavones is as high as 25% byweight or more of the total solid content; (2) the content of malonylisoflavone glycosides is 15 to 95% by weight, more preferably 30 to 90%by weight, still more preferably 35 to 85% by weight, most preferably 35to 80% by weight, by taking the sum of the total amounts of isoflavonesand saponins as 100% by weight; (3) the content of isoflavones otherthan malonyl isoflavone glycosides is 0 to 50% by weight, morepreferably 0 to 45% by weight, still more preferably 0 to 40% by weight,most preferably 0 to 35% by weight, by taking the sum of the totalamounts of isoflavones and saponins as 100% by weight; and (4) thecontent of saponins is 5 to 60% by weight, more preferably 10 to 50% byweight, still more preferably 15 to 45% by weight, most preferably 20 to40% by weight, by taking the sum of the total amounts of isoflavones andsaponins as 100% by weight.

Example 2

In 100 ml of water, 5 g of the isoflavone-containing compositionobtained in Example 1 was dissolved, and the solution was adjusted to pH3.0 with citric acid. After allowing the solution to stand overnight atroom temperature, the solution was centrifuged at 3000 g for 10 minutesand the supernatant was collected. The supernatant was adjusted to pH6.5 with sodium hydrogen carbonate, dried and powdered to obtain 4 g ofpowder. The isoflavone concentration of the isoflavone-containingcomposition thus obtained was 37% by weight of the total solid content.The isoflavone-containing composition thus obtained had extreme highsolubility in the neutral range (pH 7.0) and even in the acid range (pH3.0), which is at least 100 mg/100 ml based on the amount ofisoflavones. In addition, no precipitate and no turbidity were observedin the composition after cold storage for 2 weeks. Therefore, anisoflavone-containing composition having high solubility and goodstability under cold storage was obtained.

Example 3

Isoflavone-containing Beverage

The isoflavone-containing composition obtained in Example 1 wascompounded using the combination ratio shown in Table 6, and then UHTsterilized to prepare a beverage.

TABLE 6 Isoflavone-containing composition 0.17 g Sugar 16.0 g Grapefruitjuice 1.3 g Acidulant 1.0 g Calcium lactate 0.8 g Vitamin D3 100 IUGrapefruit flavor 0.1 g Water 80.63 g Total 200.0 g

The beverage had high solubility, good appearance and good flavor.Drinking 50 mL of the beverage can provide intake of 40 mg ofisoflavones.

Example 4

Production of Isoflavone-containing Portion Beverage

The isoflavone-containing composition obtained in 10 Example 2 wascompounded using the combination ratio shown in Table 7, sterilized andthen aseptically packed to produce portions (10 g×15).

TABLE 7 Isoflavone-containing composition 1.5 g Sugar 40.0 g Fruit juice10.0 g Citric acid 5.0 g Flavor 0.5 g Water 94.5 g Total 150.0 g

The portion thus obtained had good appearance, and had good flavor whenthe portion was diluted with 100 ml of water. Consuming one pack of thisportion can provide intake of 40 mg of isoflavones. The portion is alsoexcellent in portability.

INDUSTRIAL APPLICABILITY

According to the present invention, application of isoflavones can beremarkably expanded in all kinds of foods including beverages in whichisoflavones are required to be readily soluble in water even at lowtemperature, as well as in pharmaceuticals and cosmetics. Particularly,the present invention can greatly contribute to industry relating tohealth foods in which various physiological functions of isoflavones areutilized.

1. An isoflavone-containing composition which comprises 15 to 95% byweight of malonyl isoflavone glycosides, 0 to 50% by weight ofisoflavones other than malonyl isoflavone glycosides and 5 to 60% byweight of saponins, by taking the sum of the total amounts ofisoflavones and saponins in said composition as 100% by weight, whereinthe proportion of group A saponins in the saponins is 55% by weight ormore, by taking the total amount of saponins in the composition as 100%by weight, and the composition contains isoflavones in an amount of 25to 95% by weight of the total solid content.
 2. Theisoflavone-containing composition according to claim 1, which isprepared from soybean hypocotyls as the starting material.
 3. Theisoflavone-containing composition according to claim 1, which has awater solubility of at least 20 mg/100 ml at 25° C. based on the amountof isoflavones.
 4. A process for producing the isoflavone-containingcomposition according to claim 2, which comprises the steps of: (A)extracting soybean hypocotyls with 15 to 95% by volume aqueous ethanolat 10 to 50° C. to obtain an extract; (B) bringing a solution of theextract obtained by the step (A) in water into contact with a nonpolaradsorbent resin to allow isoflavones to be adsorbed to the resin; and(C) eluting isoflavones from the adsorbent resin with 15 to 40% byvolume aqueous ethanol.
 5. An edible composition containing theisoflavone-containing composition according to claim
 1. 6. A process forfractionating isoflavones and saponins, which comprises extractingsoybean hypocotyls with 15 to 95% by volume aqueous ethanol at 10 to 50°C. to obtain an extract, bringing a solution of the extract in waterinto contact with a nonpolar adsorbent resin, eluting anisoflavone-containing fraction from the adsorbent resin with 15 to 40%by volume aqueous ethanol, and then eluting a saponin-containingfraction from the adsorbent resin with 65 to 90% by volume aqueousethanol.